BY FUNGI

D. GOTTLIEB Department of Plant Pathology, University of Illinois, Urbana, lllinois, U.S.A.

To describe adequately the carbohydrate of the more than forty thousand species of fungi, from many diverse ecological habitats is, at this time, an impossible task. The morphology of the different species varies greatly; they range from relatively simple one-celled yeasts to the thread-like hyphal forms of the molds which ramify through and over the substrates, and to even more complex forms, such as mushrooms, which have large fruiting bodies. Despite the organization of cells into hyphae, mycelial mats, or reproductive organs, the fungi are essentially unicellular. Any single carries on all the basic metabolic functions and will give rise to all other morphological, communal, or reproductive forms of the species. Despite taxonomic sturlies of the large number of species, the biochemistry of very few üf these organisms has been studied. At best, one can describe the meta­ bolic activity of only those species that have been the object of research be­ cause of .)ome unique chemical characteristic, the~r economic importance or ease of handling. Even different members of the same species can vary greatly and the description of one strain of a species might not apply to other strains. N evertheless, we are being increasingly reassured of the funda­ mental similarity in metabolic activity among the fungi as the number of sturlies o:e these microbes steadily increases. The basic biochemical path­ way.; are those already described for most heterotrophic organisms that reqt:.ire m· can use exogenaus carbohydrate-either as an energy source or to furnish the carbon skeleton for other functional or structural metabolites. Only the hyphal fungi will be discussed in this paper, since the activities of the yeast jungi have been adequately described1. Fungi grow in or on a great number of living or non-living organic sub­ strates. Among these microbes are saprophytes, obligate parasites, and a large number of facultative parasites which can grow on either dead and living materials. The fungi are, therefore, confronted with a variety of , and the versatility of an organism in beingable to utilize a wide nurnber of such compounds could determine its chance of survival. Simple sugars, , trisaccharides and glycosides of hexoses with other types of compounds are met; but probably the carbon sources in greatest abundance are the polymeric forms of simple sugars. These poly­ saccharides must first be hydrolysed to simple sugars before further meta­ bolism occurs. Most hydrolytic in fungi are constitutive, but some­ times they are adaptive, being synthesized only in the presence of the substrate. The fungi commonly contain sucrase or other ß-fructofuranosi• dases, though a few exceptions have been noted. This sugar is usually assumed w be hydrolysed outside the cell, but recent studies have shown 603 D. GOTTLIEB the Rhiz:,octonia solani hydrolyses the sucrase at the cell surface or inside the cell2• Spores of Myrothecium verrucaria, however, are believed to metabolize sucrase without prior hydrolysis3. Maltase is also ubiquitous among the fungi, though again a few exceptions have been reported. Galactosidase, cellobiase, trehalose melibiase also occur in many of these microbes. These enzymes need not be specific for each substrate, even though tri- and tetra­ saccharides can be used. Cochrane4 pointsout that probably many of these hydrolyses are carried on by more general enzymes, such as transfructisi­ dases, ß-glucosidases, or transglycosidases. Trisaccharides can thus be split wherever the appropriate linkages occur. Starchis hydrolysed by amylase, an which has been reported in almost all species. Wolf and Wolf5 have compiled a list of the enzymes found in 23 wood-inhabiting fungi, all ofthem produced amylase. Aspergillus oryzae and A. niger are used in the commercial production of these enzymes. Most data agree that mainly the ~-amylase is made by these microbes. Crystalline amylases have been obtained from Rhizopus delemar and A. niger. is also utilized by many fungi which produce cellulase, ß-1,4- glucosidase, and hydrolyse this polymer to . A complex of enzymes is probably involved. One of these is responsible for the swelling of cellulose without the appearance of free reducing groups-perhaps by attacking rela­ tively few points of attack along the chain. The other enzyme completes the hydrolysis into cellobiose or glucose units. Gellobiase is involved in final hydrolysis to simple hexose. A cellulase in Merulius lachrymans seems to split the cellulose molecule near its centre6, 7. The cellobiase is a hydrolase with transglycosidase properties. Experiments on Myrothecium verrucaria have given different results depending on the investigators: some hold to the con­ cept of multi-component whereas in others a single enzyme has been found. Enzymes für other types of glucose polymersarealso present. Laminarirr is hydrolysed by a ß-n-1,3 glucosidases. Trehalase, ß-1,2-glucosidase, and ß-galactosidases have been reported 9. Pentosans are also hydrolysed by a number offungilO, 11. Fungiare primarily aerobic organisms, nevertheless the anaerobic Embden MeyerhofPathway (E.M.P.) occurs in almost all species. The entire E.M.P. or parts of the system are even active under aerobic condition, but reduced concentration or the absence of oxygen favour the operation of this pathway. Many fungi produce and , or often lactic acid12 and their formation from glucose strongly indicates an operative E.M.P. Among the many molds producing ethanol are A. niger, A. clavatus, A. glaucus, Mucor muceda and Rhizopus sp. That the E.M.P. is the pathway of alcohol production is shown by the 1 :1 ratio of carbon dioxide to ethanol that was produced by living cells of Fusarium lycopersici13 and F. lini14. Cell­ free preparations of A. nigeralso give similar results. Phycomycetes tend to produce more commonly than do the higher fungi. The formation of both end products involve the E.M.P. and a reduction of or by reduced nicotinamide-adenine dinucleotide (NAD) and their respective dehydrogenases. The biochemical Iiterature on fungi con­ tains many examples ofindividual reaction product that involve the E.M.P.; M. plumbus and A. niger convert pyruvate to acetaldehyde. Many wood­ rotting Basidiomycetes convert ethanol to acetaldehyde15, 16, 604 CARBOHYDRATE CATABOLISM BY FUNGI The presence of the individual enzymes in the E.M.P. have been demon­ strated ;.n cell-free preparations for A. niger17, Penicillium chrysogenumlS and Claviceps purpurea19. They are present in spores of P. oxalicum, Ustilago maydis, Puccinia graminis tritici and Uromyces phaseoli20. Evidence for the participation of this pathway is also based on the dissimilation of 14C-labelled sugars. Under anaerobic conditions, glucose-1-14C is converted by F. lini to methyl­ labelled ethanol21. In other experiments with the same species, glucose- 1-14C was dissimilated with a recovery of only 1·8 per cent of the total activity in the carbon dioxide, but with glucose-3,414C the recovery was 53·1 per cent14. These results would be expected if the E.M.P. were used as the . Under aerobic conditions, very slight conversion occurred via this system. Other evidence for the E.M.P. is the presence of the various intermediary products. P. chrysogenum mycelium contains glucose- 1-phosphate, glucose-6-phosphate, -6-phosphate, fructose-1 ,6- diphosphate, adenosirre triphosphate (ATP) and nicotinamide-adenine dinucleotide phosphate (NADP). Studies with Iactate formation from glucose-l-14C gave the correct labeHing for the system, methyl-14C Iactate and smaller amounts of methyl-14C ethanol12. The aerobic, hexose monophosphate pathway (H.M.P.) is also wide­ spread in the fungi. Like the E.M.P., it converts glucose to metabolic inter­ mediates which can later be more fully oxidized in the energy producing cydes. This system is also a good source for the pentose needed in nucleotide synthesü.. Illustrative evidence for the operation of the H.M.P. is readily available. P. chrysogenum oxidizes C-1 of glucose more readily than the other carbons. Such data indicate the initial conversion of glucose to glucose-G-phosphate, 6-phosphogluconolactone, 6-phosphogluconate; then decarboxylation to carbon dioxide and ribulose-5-phosphate22. These results were confirmed by Heath and Koffier23. They demonstrated the participation of the Zwischenferment and NADP. Active participation of the H.l\1.P. was also demonstrated by radiorespirometry techniques in Ustilago maydis and Tilletia contraversa24. The individual enzymes of the entire system were shown 1n P. chrysogenum25, Claviceps purpurea19, Puccinia graminis tritici, Uromyces phaseoli, P. oxalicum and Ustilago maydis20. One should emphasize that under aerobic conditions, both the E.M.P. and H.l\1.P. may be active simultaneously. The quantitative röle of each system could depend on the age, on morphologic development ofthe fungus26 or even nutritive conditions. A survey of the data indicates that no one pa,:hway predominates in the fungi as a group. Increasing anaerobiosis tends to favour the involvement of the E.M.P., thus, though Verticillium alhoatrurr.: uses both pathways in air, only the E.M.P. operates under anaerobic conditions. Caldariomyces fumago does not seem to use the E.M.P. though it is likely that the system is present26. Less data is available on the r6le of Entner-Doudoroff pathway (E.D) 27. The usual initial phosphorylation of glucose is not needed. Gluconic acid is J:irst formed, then phosphorylated. The molecule is further oxidized to 2-keto-3 .. deoxy-6-phosphogluconate. Aldolase splits this intermediate to pyruvic acid and glyceraldehyde-3-phosphate. The pathway has been demonstrated in Tilletia caries spores24 and in germinating spores of C. fumago26. 605 2U D. GOTTLIEB In some fungi, glucose cannot be metabolized directly by the pathways mentioned above and must be transformed so that it can enter into these systems. Penicillium brevi-compactum2B and A. niger29 first oxidize glucose to gluconic acid and 2-ketogluconic acid; only then can the molecule be oxidized via the H.M.P. An even more complex condition occurs in C. fumago. Glucose is oxidized to glucor..ic acid and 2-ketogluconic acid. The 6-phospho-2-ketogluconic acid is then formed; this compound is next. re­ duced to 6-phosphoglucona te by an adaptive enzyme. Only then is the sugar in the proper metabolic form to enter the same major degradative pathways. Further metabolism occurs via both the E.D. and H.M.P.26. For , preliminary transformations to readily oxidized sugars can be postulated. Polyporus circinatus oxidizes this hexose directly to galactonic acid by a flavoprotein system that resembles glucose oxidase and forms hydrogen peroxide30, Little information is available on the mechanism of utilization of pentoses, though many fungi use these sugars. It is obvious that, if the pentoses could be converted to ribulose-5-phosphate or xylulose-5-phosphate, subsequent oxidation by the H.M.P. could dissimilate them. P. chrysogenum can convert o-xylose and L arabinose by first reducing them with NADH to the alcohols, followed by oxidation to ribulose and xylulose31. Oospora lactis extracts readily phosphorylates, xylulose, and ribulose to their respective 5-phosphates32. Comparatively little energy could be derived by an organism in the reactions that have metabolized glucose by the pathways previously men­ tioned. Ethanol, lactic acid, pyruvic acid, glycolaldehyde and some carbon dioxide were produced. The derivation of maximum energy from glucose requires its complete oxidation. This oxidation is accomplished by the aerobic tricarboxylic acid cycle (T.C.A.). The 2 and 3 carbon fragments from sugars are transformed into acetyl CoA which condenses with oxalo­ acetate to form citrate. By a series of metabolic transformations, the citrate loses 2 carbons as carbon dioxide to finally form again oxaloacetate. Since glucose can form 2 moles of pyruvic acid by the E.M.P., and each mole of pyruvate can be decarboxylated to form acetyl CoA, 3 cycles of the T.C.A. would account for the complete oxidation of glucose. An operative T.C.A. is probably present in all fungi. Much of the evidence is indirect and is based on the ability of fungi to oxidize the T.C.A. intermediates when the com­ pounds can permeate their cell membranes. If the membranes are imper­ meable to these exogenaus acids, cell-free preparations serve. C.fumago, for example, oxidizes citrate, ct-ketoglutarate, succinate, fumarate, and malate; similar results were found with A. niger26, Inhibitors of the various reactions in the cycle also point to the presence of the T.C.A. when such inhibitors decrease or prevent the oxidation of the substrate. These effects, if combined with oxidation pattern, have strengthened the evidence for the röle of this cycle. In the C. fumago and P. chrysogenum, oxidation of the substrateswas inhibited by fluoroacetate, arsenite, and dimethyl malonate26, 33. The presence of the enzyme that condenses acetyl CoA with oxaloacetate was demonstrated in A. niger34 andin P. oxalicum35. The T.C.A. is also present, in P. digitatum36, Uromyces phaseoli, Ustilago maydis and P. graminis tritici20. An abnormal functioning of the T.C.A. cycle is believed tobe the cause of citric acid accumulation. A. niger split glucose into two Cg fragments,. 606 CARBOHYDRATE CATABOLISM BY FUNGI presumably pyruvate. One of these pieces is decarboxylated to acetate and the other condensed with carbon dioxide by a "Wood-Werkman" reaction to yield oxaloacetate. The C 2 and C4 acids combine to form citric acid the first step in the T.C.A. cycle37. Another pathway for the net synthesis of C4 acids is by the "glyoxalate by-pass". In this reaction, acetyl CoA•eondenses normally with oxaloacetate in the T.C.A. cycle to give citrate then isocitrate. The isocitrate is not oxidizecl further, but is instead dissimilated to succinate and glyoxalate. Another acetyl CoA then combines with the glyoxalate to form malate. This cycle occurs in A. niger38, spores of P. oxalicum35, Ustilago rnaydis, Urornyces pha.seoli, and Puccinia grarninis20. Unlike many other microbes in which the "by-pass" is adaptive, this pathway is constitutive in the fungi35, 20. As indicated previously, fungi can also form C4 acids by a non-photo­ synthetic carbon dioxide fixation. The Ca for this reaction comes from the E.M.P. Cell-free preparations of A. niger fixed carbon dioxide either by condensation with phosphoenol pyruvic (PEP) acid or with pyruvic acid to give oxaloacetic acid39. The Wood-Werkman type reaction has been pre­ posed fi>r the formation of fumaric acid anaerobically by Rhizopus nigri­ cans40. Uredospores of the rust fungi, Puccinia grarninis tritici and Uromyces phaseoli can make oxaloacetate from the PEP and carbon dioxide41. The energy yielding reactions involved in the oxidation of glucose and its in1:ermediates require the movement of negative charges via the terminal electron transport system to oxygen which then combines with hydrogen ions to form water42,43, This system is present in all aerobic fungi. The first stage usually requires the removal of hydrogen from the various substrates and their transfer to NAD or NADP to form their reduced analogues. SL.ccinic acid oxidation by-passes this step to form reduced flavoprotein di;:ectly. Dehydrogenases for these reactions are widely distributed in fungi. Arecent study on ten species distributed amongst the three major groups of fungi showed the presence of the dehydrogenases for glucose-6-phosphate, triose phosphate, isocitrate, succinate, and malate. No pyruvate or keto­ glutarate dehydrogenases were detected but their absence was ascribed to the lability of these two enzymes in the fungi during preparation of the cell­ free extracts44, 45. Complete terminal electron transport systems, similar to those fo und in mammalian .cells, were presen t in all these fungi as weil as in othe~r species46, 47. Mitochondria, with which the terminal electron transport system is associated arealso present in fungi. Flavoprotein, as weil as cytochromes types a, b, and c have been demonstrated in Schizophyllum commune'l7. Cytochrome a, b, and c have been found in 45 different species of fungi48. Coenzyme Q9 and Q1o are present in Ustilago zeae and Q10 in Agaricus campestris49, Cytochrome oxidase occurs in many other fungi though the presence of complete electron transport had not been studied50-52, Certain strains of Neurospora53 and Myrothecium verrucaria54,55 have been reported to contain a cyanide-insensitive respiratory system. Fung) thus have many mechanisms by which carbohydrates can be meta­ bolized. For maximum energy production, the various pathways prepare the way for the formation ofacetyl CoASH, its entry into the and complete oxidation to carbon dioxide. The intermediary compounds that are· produced by these pathways can also be siphoned off to undergo 607 D. GOTTLIEB other reactions which are used for the structural or metabolic functions of the cell. The pentoses in the H.M.P. or oxalacetate and ~-ketoglutarate of the T.C.A. systems serve in this capacity. The use of such intermediates for synthesizing mono and polynucleotides or amino-acids depends on the needs of the cell at the time, and is governed by the relative rates of com­ peting reactions. Sugars are also utiiized in many other ways. The synthesis of ascorbic acid by fungi is a more direct conversion and does not require the catabolism of glucose. Similarly, chitin, sta-rch, or cellulose synthesis does not involve catabolic pathways. The acetyl groups that are formed by various means also need not go into the energy producing cycles; for fungi use them as a reservoir of two carbon units to build many of the essential and non-essential products of their metabolism.

References 1 A. H. Cook. The Chemistry and Biology of Yeasts, Academic Press, New York (1958). 2 M. K. Tolba and A. M. Salama. Arch. Mikrobiol. 36, 23 (1960). 3 G. R. Mandels. Plant Physiol. 29, 18 (1954). 4 W. W. Cochrane. Physiology qf Fungi, John Wiley, New York (1958). 5 F. A. Wolf and F. T. Wolf. The Fungi, Vol. II, John Wiley, New York (1947). 6 P. Kooyman. Enzymologia 18, 371 (1957). 7 J. Weigl. Arch. Mikrobiol. 38, 411 (1961). 8 B. A. Stone. Biochem. J. 66, 1P (1957). 9 R. Davies. Biochem. et Biophys. Acta 33, 481 (1959). 10 H. Lyr. Arch. Mikrobiol. 34, 189 (1959). 11 F. ]. Simpson. Can. J. Microbiol. 5, 99 (1958). 12 S. A. Waksman and J. W. Poster. J. Agr. Research 57, 873 (1938). 13 G. H. Pritham and A. K. Anderson. J. Agr. Research. 55, 937 (1937). 14 V. W. Cochrane. Mycologia 48, I (1956). 16 F. F. Nord and J. C. Vitucci. Arch. Biochem. Biophys. 9, 419 (1946). 16 F. F. Nord andJ. C. Vitucci. Arch. Biochem. Biophys. 14, 243 (1947). 17 V. Jagannathan and K. Singh. Enzymologia 16, 150 (1953). 18 C. ]. Sih and S. G. Knight. J, Bacteriol. 72, 694 (1956). 19 J. K. McDonald, V. H. Cheldelin, and T. E. King. J. Bacteriol. 80, 61 (1960). 20 P. G. Caltrider. Thesis, Illinois (1962). 21 E. C. Heath, D. Nasser, and H. Koffier. Arch. Biochem. et Biophys. 64, 80 (1956). 22 C. W. Fiebre and S. G. Knight. J. Bacteriol. 66, 170 (1953). 28 E. C. Heath and H. Koffier. J. Bacteriol. 71, 174 (1956). 24 R. W. Newburgh and V. H. Cheldelin. J. Bacteriol. 76, 308 (1958). 26 C. J. Sih, P. B. Hamilton, and S. G. Knight. J. Bacteriol. 73, 447 (1957). 26 S. Ramachandran and D. Gottlieb. Biochem. et Biophys. Acta 69, 74 (1963). 27 N. Entner and M. Döudoroff. J. Biol. Chem. 196, 853 (1952). 28 P. Godin. Antonie van Leeuwenhoek J. Microbiol. Sero!. 21, 83 (1955). 29 W. W. Cleland and M. ]. Johnson. J. Biol. Chem. 220, 595 (1956). 30 J. A. 0. Cooper, W. Smith, M. Basilla, and H. Medina. J. Bio!. Chem. 234, 445 (1959). 31 1 C. Chiang and S. G. Knight. Bacteriol. Proc. 1960, 182. 32 V. Moret and L. Sperti. Arch. Biochem. et Biophys. 98, 124 (1962). 33 E. P. Goldschmidt, I. Yall, and H. Koffier. J. Bacteriol. 72, 436 (1956). 84 C. V. Ramakrishnan and S. M. Martin. Chem. Ind. (London) 1954, 160. 35 S. Ramachandran and D. Gottlieb. Biochim. et Biophys. Acta 69, 74 (1963). 36 E. P. Noble, D. R. Reed, and C. H. Wang. Can. J. Microbiol. 4, 469 (1958). 37 W. W. Cleland and M. ]. Johnson. J. Biol. Chem. 208, 679 (1954). 38 H. L. Kornberg and J. F. Collins. Biochem. J. 68, 3P (1958). 39 C. L. Woronick and M. J. Johnson. J. Bio!. Chem. 235, 9 (1960). 4° ]. W. Foster and J. B. Davis. J. Bacteriol. 56, 329 (1948). 41 R. C. Staplesand L. H. Weinstein. Contrib. Boyce Thompson Inst. 20, 71 (1959). 42 D. E. Green. Advances in Enzymol. 21, 73 (1959). · 43 E. C. Slater. Advances in En.zymol. 20, 147 (1958). 44 W. M. Dowler, P. D. Shaw, and D. Gottlieb. J. Bacteriol. 86, 9 (1963). 46 C. Godzeski and R. W. Stone. Arch. Biochem. Biophys. 59, 133 (1955). u G. Kikuchi and E. S. G. Barron. Arch. Biochem. Biophys. 84, 96 (1959). 608 CARBOHYDRATE CATABOLISM BY FUNGI 47 :0. J. Kiederpruem and D. P. Hackett. Plant Physiol. 36, 79 (1961). 48 D. Boulter and E. Derbyshire. J. Exptl. Botany 8, 313 (1957). u R. E. Erickson, K. S. Brown, Jr., D. E. Wolf, and K. Folkers. Arch. Biochem. Biophys. 90, :n4 (1860). 50 G. S. \Vhite and G. A. Ledingham. Plant Physiol. Supple. 35, xi (1960). 51 C. J. Sth, P. B. Hamilton, and S. G. Knight. J. Bacteriol. 75, 623 (1958). 52 A. S. Sussman and C. L. Markert. Arch. Biochem. Biophys. 45, 31 (1953). 53 A. Tissieres, H. K. Mitchell, and F. A. Haskins. J. Bio!. Chem. 205, 423 (1953). 54 G. W. Kidder, III. Plant Physiol. 36: Supplement, .p. xxxii (1961). 55 R. T. Darby and D. R. Goddard. Physiol. Plantarum 3, 435 (1950).

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